ABSTRACT
In Eukaryotes, luminal and transmembrane proteins are moved to their
functional locations by conserved membrane trafficking machinery. In this process,
cargo adaptors bind motifs present on cargo, indirectly linking the proteins to coats,
which deform membranes and form transport vesicles. Here, cargo adaptor
recruitment and cargo recognition was studied by characterizing associated factors in
the budding yeast Saccharomyces cerevisiae. Possible cargo adaptor-associated
factors were identified in a proteomics study that grouped protein-protein interactions
into 501 putative membrane associated complexes using a Markov clustering
algorithm. Two clusters were selected for this work.
The first contained the uncharacterized protein Ssp120 with the endoplasmic
reticulum-to-Golgi trafficking complex Emp46/Emp47. Ssp120 stably interacted with
the Emp46/Emp47 complex and depended on Emp47 for its punctate localization.
The C-terminus of Ssp120 mediated the interaction. Homology with human MCFD2
suggests that Ssp120 may link a subset of cargo to Emp46/Emp47.
The second cluster was comprised of retromer, an endosome-to-Golgi
trafficking complex, and the Rab5-family guanine nucleotide exchange factor (GEF)
Muk1. Both Muk1 and the other known Rab5-family GEF, Vps9, interacted with
retromer and the presence of at least one was required for retromer recruitment to
endosomes. Additionally, a new VPS9 domain-containing protein present was
identified and shown to complement loss of MUK1 and VPS9. Retromer recruitment
was shown to be dependent on putative GEF catalytic residues and the presence of
their target Rabs. Furthermore, loss of GEFs resulted in mislocalization of the
potential Rab5 effector, Vps34, and its lipid product, phosphatidylinositol 3-phosphate
(PI3P), to the vacuolar membrane. As retromer is recruited by PI3P, the data support
a positive feedback model whereby retromer interacts with GEFs to indirectly modify
the lipid composition of the membrane allowing further localized recruitment.
This study validates the approach of studying novel interactors of cargo
recognition complexes to better understand their function. It suggests that Ssp120
may recognize a subset of Emp46-Emp47 cargo, indicating that an associated factor
can diversify the proteins recognized by a given cargo adaptor. Furthermore, the work
on retromer suggests a novel mechanism for the reinforcement of cargo selective
complex recruitment that may be conserved in humans.